With a worldwide production capacity of more than 100 million tons and a decreasing commodity price, methanol is regarded as a highly attractive alternative non-food raw material for biotechnology sector. The supply of methanol comes from both fossil and renewable resources, rendering it a highly flexible and sustainable raw material. C1 compounds are used by specialized groups of microorganisms i.e. the methylotrophs as their sole source of carbon and energy. While progress to use natural methylotrophs in biotechnology is on-going, ECOMUT propose to launch an alternative strategy by integrating methylotrophy into the established bacterial production host Escherichia coli. The synthetic biology approach we plan will be combined with a systems level understanding of methylotrophy. This will not only generate new a fundamental knowledge of C1 metabolism but will also provide a biological platform capable of transforming methanol in any molecule of interest. This research will make a significant contribution towards unleashing the potential of methanol as a raw material in a vast range biotechnological applications in any industrialised location.

The performance of many processes such as sewage treatment plants is highly related to the oxygen transfer from air bubbles, usually injected through diffusers, to microorganisms able to degrade pollutants contained in wastewaters. However, characterizing accurately the oxygen mass transfer in such processes is still a challenging issue mainly because of the liquid phase complexity. The aim of this project is to develop specific techniques and rigorous models to better estimate the various mechanisms that locally govern the gas/liquid mass transfer process. New and efficient visualization techniques are proposed to visualize and estimate the surfactant migration, structure and layer thickness. A new modelisation will permit to adapt and reduce the energy consumption of aeration that represents 80% of the global process energy.

This project aims at studying the role of gene expression variability (due to the stochastic fluctuations at the molecular level) in stress response and genetic instability. The impact of this variability on population dynamics is now well-studied, and increase of stochasticity (or noise) in gene expression is considered as a relevant evolutionary strategy in fluctuating environments. Here we want to determine if such an increase for some genes has been a way for technological yeast strains to adapt to the stressful fluctuating conditions they have to deal with. Indeed these strains are well-adapted to many environmental stress compared to laboratory strains. In the first part of this project, we will focus on the recently sequenced oenological strain of Saccharomyces cerevisiae EC1118 (NOVO et al. 2009) to detect promoters that are noisier in this strain compared to the standard non-adapted laboratory strain S288c. If such differences of noise are detected, we will study their impact on stress response and adaptation in stressful environments, especially in terms of fitness. This original stragety should enable the identification of new determinants of stress resistance and tolerance. At the moment no study has linked noise in gene expression to genetic variability. But, like any other phenotype, maintenance of genome integrity is under the influence of genes expressed with stochastic fluctuations. The rate of genetic-variant generation (RGVG) could be variable as a consequence of stochastic fluctuations in the expression of DNA repair and maintenance genes from cell-to-cell. High noise in the expression of genes involved in Double-Strand Break repair or DNA replication for instance, could confer a broad range RGVG from cell-to-cell in the population, and favour the emergence of sub-populations with higher genetic variability in times of stress, thanks to a second-order selection process (indirect selection of mutator strains along with favourable mutations they generate which counterbalance possible deleterious mutations) (CAPP, 2010). The aim of this project is to determine if industrial strains have evolved towards such a high noise in the expression of genes involved in DNA repair and maintenance. If this is the case, we will study the impact of different noise levels in the xepression of these genes on genetic variability under selective conditions. Capp, J. P. (2010). Noise-driven heterogeneity in the rate of genetic-variant generation as a basis for evolvability. Genetics 185, 395-404 Novo, M., et al. (2009). Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118. Proc Natl Acad Sci U S A 106, 16333-16338.

In the period of unprecedented expansion of energy demand and the desire to reduce greenhouse gas emissions, industrial biotechnology is expected to provide solutions to contribute to a more sustainable society, using for example renewable resources and waste as raw materials for manufacturing of bulk and fine chemicals and energy. By their abilities to combine plants properties (CO2 as substrate for the production of complexes molecules) and microorganism’s properties (rapid growth), microalgae are undoubtedly attractable for transition towards renewable energy. Despite of the high biotechnological potential of microalgae in nutrition, cosmetic, nutraceutical markets, there are a number of barriers to overcome to make them economically viable for mass markets such as energy and green chemistry. To meet this challenge, several national and european initiatives aim at creating a partnership between academic scientific communities and industry in which industrial specifications and constraints are taking into account by academics to identify leverage actions to enhance the competitiveness. The SynDia project in line with this approach will generate considerable added value and provide the necessary impetus to significantly accelerate the development of industrial biomanufacturing processes. My double experience in academic laboratories and in a biotechnology company enables me to structure my research around the development of knowledge and methodologies to circumvent industrial bottlenecks. Synthetic biology is emerging as an important sub-area of industrial biotechnology. This new field deals with the development of biocatalysts using an engineering approach to both improve productivity of natural compounds and design and construct novel biological parts, devices and systems to perform new functions. Synthetic biology requires the creation of microalgae chassis platform, robustness in challenging processes straightforward genome engineering and with an efficient regulatory structure. The diatom, Phaeodactylum tricornum is one of them. This species able to produce huge amount of lipids is already exploited for the production of long chain fatty acid such as EPA. Regarding the metabolic engineering of Phaeodactylum tricornutum, the production of engineered strain is still far from straightforward process and there is a need for faster and more effective genome engineering methodologies. In this research program we plan to develop microalgae as industrial biocatalysts for the production of fuels and chemical and to achieve this goal, we propose: 1. To develop a new class of genome-specific nucleases and modulate double-strand break mechanisms in order to achieve improved genome modification frequencies adapted to study of gene function and/or to create strains with novel genetic properties 2. To identify optimal loci suitable for transgene expression in order to ensure the efficacy of their expression in terms of level as well as stability over time and to maximize the safety of genome editing 3. To generate artificial transcriptional modulators and synthetic promoters which will provide the means to tune gene expression in metabolic pathways and thus strains cable of high efficiency conversion of natural resources into target industrial commodities 4. To exemplify the power of the genomic tools to harness P. tricornutum for biofuel production by engineering lipid metabolism quantitatively as well as qualitatively Altogether, SynDia will deliver a microalgae platform adapted for the industrial production, leading microalgae among the key enablers of the bioeconomy transition. This project will open new doors for biotechnological applications, notably as regards to the reengineering of diatoms’ lipid metabolism for biofuel production and the creation of artificial metabolic pathway for energy and green chemistry.

Polysaccharides and oligosaccharides are molecules of interest for many types of applications in Food and Feed (e.g. prebiotics, additives), Agricultural (e.g. fertilizers), Health (e.g. vaccine, antibiotics, antivirals, biomaterials) as well as Bulk and Fine Chemicals (e.g. bioplastics, biosurfactants) applications. However, their complex structures make them difficult to synthesise by traditional chemistry. Bioprocesses using enzymes or cells have demonstrated their superiority for efficient and eco-friendly production of such compounds. In addition, progress in Synthetic Biology and Metabolic Engineering nowadays offers new opportunities for microbial engineering and design aiming at improving the synthetic utility of microorganisms. In this context, the ENGEL project will focus on the engineering of a microbial strain, natural producer and secretor of sucrose-active enzymes, in order to deliver a microbial platform adapted for the rapid industrial production of these enzymes and tailor made polysaccharides, oligosaccharides and glyco-conjugates. Low production levels, tedious purification, and unfavourable environmental footprint limit the developments of the applications of such enzymes and biopolymers. The use of recombinant enzymes (mainly produced by E. coli) that could circumvent such drawbacks is even more delicate, as E. coli cannot secrete high molecular weight enzymes. The leading goal of the ENGEL project is the demonstration that natural microbial strains can be tuned on purpose to serve as an efficient platform for the production of innovative tailor-made glucoderivatives. To allow metabolic engineering of such strains, molecular tools will be specifically established using a cutting edge genomic technology based on specifically designed nucleases. The technology will be applied for the first time to the industrially-relevant selected species, known to be natural producer of sucrose-active enzymes and polysaccharides. The program will be directed to ensuring the versatile use of the chassis cell for i) highly efficient production of different sucrose-active enzymes, and ii) highly efficient in vivo expression of different sucrose-active enzymes, thus allowing one pot fermentation production process of structurally controlled polymers showing biophysical properties of interest for various usages. The research efforts should allow new, sustainable and economical integrated production concept to be developed. A particular attention will be dedicated to the respect of Green Chemistry rules allowing energy input reduction and reduced toxic reagent utilization for product recovery. Substantial advances on engineering of the selected strain and breakthrough solutions for enzyme and polymer production should accelerate the eco-friendly manufacturing of the target compounds and reduce their production costs. It is expected that the panel of products that could be produced thanks to the engineered cellular platform will be enlarged, thus facilitating the access to new markets.